Bottom Line:
However, cancer cells often develop resistance to cisplatin, which limits therapeutic effectiveness of this otherwise effective genotoxic drug.This unexpected finding involves a switch from the G2/M to G1 checkpoint accompanied by decrease in ATM/Chk2 and increase in ATR/Chk1 phosphorylation.In conclusion, inhibition of ERβ considered as a supplemental anticancer therapy, has been found to interfere with cisplatin-induced cytotoxicity in human medulloblastoma cell lines.

ABSTRACTCisplatin is one of the most widely used and effective anticancer drugs against solid tumors including cerebellar tumor of the childhood, Medulloblastoma. However, cancer cells often develop resistance to cisplatin, which limits therapeutic effectiveness of this otherwise effective genotoxic drug. In this study, we demonstrate that human medulloblastoma cell lines develop acute resistance to cisplatin in the presence of estrogen receptor (ER) antagonist, ICI182,780. This unexpected finding involves a switch from the G2/M to G1 checkpoint accompanied by decrease in ATM/Chk2 and increase in ATR/Chk1 phosphorylation. We have previously reported that ERβ, which is highly expressed in medulloblastomas, translocates insulin receptor substrate 1 (IRS-1) to the nucleus, and that nuclear IRS-1 binds to Rad51 and attenuates homologous recombination directed DNA repair (HRR). Here, we demonstrate that in the presence of ICI182,780, cisplatin-treated medulloblastoma cells show recruitment of Rad51 to the sites of damaged DNA and increase in HRR activity. This enhanced DNA repair during the S phase preserved also clonogenic potential of medulloblastoma cells treated with cisplatin. In conclusion, inhibition of ERβ considered as a supplemental anticancer therapy, has been found to interfere with cisplatin-induced cytotoxicity in human medulloblastoma cell lines.

pone-0033867-g004: Inhibition of ERβ modulates cisplatin-induced phosphorylation of cell cycle checkpoint proteins.Panel A: Western blot analyses showing levels of the phosphorylated ATM, ATR, Chk1 and Chk2 in constitutively growing Daoy cells (10%FBS) treated with cisplatin (1 µg/ml) in the presence (Cis+ICI) or absence (Cis) of ICI182,780 (10 µM). The cells without treatment (FBS), or cells treated with ICI182,780 only (ICI) were used as controls. Panel B: Densitometry of Western blots depicted in Panel A evaluated by EZQuant-Gel 2.17 software. Levels of pATM, pATR, pChk1 and pChk2, were normalized with the corresponding levels of Grb-2. Data represent averages obtained from densitometric measurements of 3 blots with standard deviation and each band was normalized with corresponding loading control, Grb-2.

Mentions:
If indeed this transition in cell cycle distribution is based on DNA damage/cell cycle checkpoint system, we should observe also a shift in the phosphorylation pattern between ATM/Chk2 and ATR/Chk1 [22]. Of note, the cisplatin treatment is expected to trigger G2/M arrest followed by elevated apoptosis [23]. The results in Fig. 4 demonstrate very low levels of phosphorylation of ATM, ATR, Chk1 and Chk2 in the absence of DNA damage (FBS and ICI). Following the treatment with cisplatin (Cis), over 4-fold increase in ATM/Chk2 phosphorylation and 1.4-fold increase of ATR/Chk1 phosphorylation were observed after 6 hours. The phosphorylation pattern between ATM/Chk2 and ATR/Chk1 was reversed when the cisplatin treated cells were compared to the cells treated with cisplatin + ICI182,780. Quantitatively, ATM/Chk2 phosphorylations decreased by an average of 2-fold and ATR/Chk1 phosphorylations increased by an average of 1.5-fold (Fig. 4B). These results demonstrate that the transition from G2/M to G1 arrest observed in the presence of ICI182,780 was indeed accompanied by the transition from ATM/Chk2 to ATR/Chk1 activation.

pone-0033867-g004: Inhibition of ERβ modulates cisplatin-induced phosphorylation of cell cycle checkpoint proteins.Panel A: Western blot analyses showing levels of the phosphorylated ATM, ATR, Chk1 and Chk2 in constitutively growing Daoy cells (10%FBS) treated with cisplatin (1 µg/ml) in the presence (Cis+ICI) or absence (Cis) of ICI182,780 (10 µM). The cells without treatment (FBS), or cells treated with ICI182,780 only (ICI) were used as controls. Panel B: Densitometry of Western blots depicted in Panel A evaluated by EZQuant-Gel 2.17 software. Levels of pATM, pATR, pChk1 and pChk2, were normalized with the corresponding levels of Grb-2. Data represent averages obtained from densitometric measurements of 3 blots with standard deviation and each band was normalized with corresponding loading control, Grb-2.

Mentions:
If indeed this transition in cell cycle distribution is based on DNA damage/cell cycle checkpoint system, we should observe also a shift in the phosphorylation pattern between ATM/Chk2 and ATR/Chk1 [22]. Of note, the cisplatin treatment is expected to trigger G2/M arrest followed by elevated apoptosis [23]. The results in Fig. 4 demonstrate very low levels of phosphorylation of ATM, ATR, Chk1 and Chk2 in the absence of DNA damage (FBS and ICI). Following the treatment with cisplatin (Cis), over 4-fold increase in ATM/Chk2 phosphorylation and 1.4-fold increase of ATR/Chk1 phosphorylation were observed after 6 hours. The phosphorylation pattern between ATM/Chk2 and ATR/Chk1 was reversed when the cisplatin treated cells were compared to the cells treated with cisplatin + ICI182,780. Quantitatively, ATM/Chk2 phosphorylations decreased by an average of 2-fold and ATR/Chk1 phosphorylations increased by an average of 1.5-fold (Fig. 4B). These results demonstrate that the transition from G2/M to G1 arrest observed in the presence of ICI182,780 was indeed accompanied by the transition from ATM/Chk2 to ATR/Chk1 activation.

Bottom Line:
However, cancer cells often develop resistance to cisplatin, which limits therapeutic effectiveness of this otherwise effective genotoxic drug.This unexpected finding involves a switch from the G2/M to G1 checkpoint accompanied by decrease in ATM/Chk2 and increase in ATR/Chk1 phosphorylation.In conclusion, inhibition of ERβ considered as a supplemental anticancer therapy, has been found to interfere with cisplatin-induced cytotoxicity in human medulloblastoma cell lines.

ABSTRACTCisplatin is one of the most widely used and effective anticancer drugs against solid tumors including cerebellar tumor of the childhood, Medulloblastoma. However, cancer cells often develop resistance to cisplatin, which limits therapeutic effectiveness of this otherwise effective genotoxic drug. In this study, we demonstrate that human medulloblastoma cell lines develop acute resistance to cisplatin in the presence of estrogen receptor (ER) antagonist, ICI182,780. This unexpected finding involves a switch from the G2/M to G1 checkpoint accompanied by decrease in ATM/Chk2 and increase in ATR/Chk1 phosphorylation. We have previously reported that ERβ, which is highly expressed in medulloblastomas, translocates insulin receptor substrate 1 (IRS-1) to the nucleus, and that nuclear IRS-1 binds to Rad51 and attenuates homologous recombination directed DNA repair (HRR). Here, we demonstrate that in the presence of ICI182,780, cisplatin-treated medulloblastoma cells show recruitment of Rad51 to the sites of damaged DNA and increase in HRR activity. This enhanced DNA repair during the S phase preserved also clonogenic potential of medulloblastoma cells treated with cisplatin. In conclusion, inhibition of ERβ considered as a supplemental anticancer therapy, has been found to interfere with cisplatin-induced cytotoxicity in human medulloblastoma cell lines.